Amyloid protein accumulation is a histopathological change occurring in cerebral tissues of not only those with Alzheimer's disease or Down's syndrome but also those who went through a process of normally aging. The amyloid protein, which consists of from 40 to 42/43 amino acids rich in hydrophobic ones, is produced from its precursor, the amyloid-precursor protein (hereinafter referred to as APP), by a hydrolytic cleavage. APP, which consists of 695, 751 or 770 amino acids, is a type 1 membrane protein that spans the membrane once, with its amino terminal portion exposed outside the cell. The difference in the number of its amino acids corresponds to the presence or absence of a so-called Kunitz-type protease inhibitor-active site, which is located in the region outside the cell. In neurocytes, a form of APP consisting of 695 amino acids (hereinafter referred to as “APP695”, whose amino acid sequence is set forth as SEQ ID NO:7), is a dominant one. The APP695, consisting of 695 amino acids starting with the methionine at position 1 and extending up to the asparagine at position 695, is an isoform occurring mainly in neurocytes, and is a type 1 membrane protein having a transmembrane domain (consisting of 24 amino acid extending from the glycine at position 625 to the leucine at position 648) which spans the cell membrane once. The amyloid protein comprises a shorter form protein molecule consisting of 40 amino acids extending from the aspartic acid at position 597 in the APP695's portion exposed outside the cell to the valine at position 636, which is within the cell membrane, and a longer protein molecule consisting of 42 or 43 amino acids extending up to the alanine at position 638 or the threonine at position 639.
On the other hand, a form of APP consisting of 770 amino acids (hereinafter referred to as “APP770”, whose nucleotide sequence in the coding region is set forth as SEQ ID NO:8 and whose amino acid sequence as SEQ ID NO:9) is an APP gene product consisting of 770 amino acids extending from the methionine at position 1 to the asparagine at position 770 and includes in it an amino acid sequence portion that the APP695 does not possess (75 amino acids extending from the glutamic acid at position 289 to the lysine at position 363). Apart from this inserted amino acid sequence, it is the molecule having exactly the same amino acid sequence as those of APP695. Besides the APP695 and the APP770, there is an isoform called APP751, which consists in total of 751 amino acids, with 19 amino acids lost that extends from the methionine at position 345 to the lysine at position 363 of the APP770. An amino acid sequence that is commonly inserted into APP751 and APP770 (56 amino acids extending from the glutamic acid at position 289 to the alanine at position 344 in the APP770 amino acid sequence) possesses an activity of the Kunitz-type protease inhibitor and is thought to be expressed in cells other than neurocytes.
Since a hypothesis was experimentally proven that toxicity on neurocytes is one of the physiological activities of the amyloid protein [Yankner,-B-A; Dawes,-L-R; Fisher,-S; Villa-Komaroff,-L; Oster-Granite,-M-L; Neve,-R-L. Neurotoxicity of a fragment of the amyloid precursor associated with Alzheimer's disease. (1989) Science. 245(4916): 417-20, and Yankner,-B-A; Duffy,-L-K; Kirschner,-D-A. Neurotrophic and neurotoxic effects of amyloid beta protein: reversal by tachykinin neuropeptides. (1990) Science. 1990 250(4978): 279-82], it has been assumed as a key molecule of the onset of Alzheimer's disease. Two important steps of reactions must take place for the amyloid protein to be produced from the amyloid-precursor protein. The first step is the cutting off, by β-secretase, of the portion extending from the amino terminus of the amyloid protein portion. At the second step, cleavage takes place at the carboxyl terminus of the amyloid protein by the action of γ-secretase, resulting in the release of the amyloid protein, with dissociated APP intracellular fragment left behind. According to conventional findings, the cleavage in the second step has been assumed to occur at the gamma (γ) site, which is at the carboxyl terminus of the amyloid protein (between the valine at position 711 and the isoleucine at position 712, between alanine at position 713 and the threonine at position 714, or between the threonine at position 714 and the valine at position 715, according to the manner of numbering of amino acid residues in the APP770). In recent findings, however, it is reported that the cleavage occurs at the upsilon (ε) site, downstream by further 5-10 amino acid residues (in the direction of the carboxyl terminus), and thus more close to the cytoplasm (between the threonine at position 719 and the leucine at position 720, or between the leucine at position 720 and the valine at position 721, according to the manner of numbering of amino acid residues in the APP770).
In Alzheimer's disease, neuronal lesions in the brain occur before its abnormal clinical symptoms will appear, such as disorientation, debilitating memory loss, amnesia, deteriorating judgment, and behavioral aberration. The neuronal lesions include deposition of the amyloid protein, neurofibrillary tangle, and degenerative cellular loss, among which deposition of the amyloid protein is the earliest pathological event.
The amyloid hypothesis, according to which the production and deposition of amyloid protein does cause the disease, is deemed important with respect to the progress of Alzheimer's disease. The basis for it has been provided by studies of familial Alzheimer's disease.
There is a speculation yet to be confirmed that γ-secretase is the gene product of presenilin-1 [Sherrington,-R; Rogaev,-E-I; Liang,-Y; Rogaeva,-E-A; Levesque,-G; Ikeda,-M; Chi,-H; Lin,-C; Li,-G; Holman,-K; et-al. Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease. (1995) Nature, 375(6534), 754-760] and presenilin-2 [Levy-Lahad,-E; Wasco,-W; Poorkaj,-P; Romano,-D-M; Oshima,-J; Pettingell,-W-H; Yu,-C-E; Jondro,-P-D; Schmidt,-S-D; Wang,-K; et-al. Candidate gene for the chromosome 1 familial Alzheimer's disease locus. (1995) Science, 269(5226), 973-977, and Rogaev,-E-I; Sherrington,-R; Rogaeva,-E-A; Levesque,-G; Ikeda,-M; Liang,-Y; Chi,-H; Lin,-C; Holman,-K; Tsuda,-T; et-al. Familial Alzheimer's disease in kindreds with missense mutations in a gene on chromosome 1 related to the Alzheimer's disease type 3 gene, (1995) Nature 376(6543), 775-778], which were found on the chromosome 14 as causative genes of early-onset familial Alzheimer's disease.
That the APP is also a causative gene of early-onset familial Alzheimer's disease (Goate, A. et al Nature 1991) indicates that a plurality of genes are involved as causative factors of the single class of dementia called Alzheimer's disease.
As mentioned hereinbefore, the amyloid protein comprises two components. One is a shorter amyloid protein component, which starts with aspartic acid and ends in the valine at position 40, and the other is a longer amyloid protein component consisting of 42 or 43 amino acids, which starts with the same aspartic acid as does the 40 amino-acids component but ends in the alanine at position 42 (thus longer by 2 amino acid residues) or the threonine at position 43 (thus longer by 3 amino acid residues). The latter longer component is more hydrophobic and therefore still less soluble. Through addition of the shorter components around the longer one serving as a core, amyloid fiber is formed, which is a fibriform structure having the diameter of about 5 to 6 nm.
It has been demonstrated that the production of the longer amyloid protein increases where a genetic mutation occurs in presenilin-1 or presenilin-2. It is thus thought that this effect of mutation causes elevated formation of the longer amyloid protein component, which assumedly determines the threshold level of polymerization of the amyloid protein, and thereby accelerates the pathogenic reaction.
A number of genetic mutations in the APP have been identified, typical ones of which are roughly divided into Sweden mutation (Met670Asn, Lys671Leu, according to the manner of numbering of amino acid residues in the AP770. The same applies also to mutations described below.), Dutch-type mutation (Glu693Gln), London mutation (Val717Ile), and Australia mutation (Leu723Pro), which are located just before the position corresponding to the amino terminus of amyloid protein. While production of both components of the amyloid protein are found to increase in Sweden mutation, only the production of the longer amyloid protein component increases in London mutation and Australia mutation. The effect of Dutch-type mutation is still under discussion and no conclusion has been reached.
There is an allele E4, a risk factor relating to apolipoprotein E. Statistical analyses demonstrated that the onset of Alzheimer's disease will accelerated by 8 to 10 yeas where only one allele on the pair of chromosomes is E4, and by 16 to 20 years where the alleles are E4/E4 on the both chromosomes [Corder,-E-H; Saunders,-A-M; Strittmatter,-W-J; Schmechel,-D-E; Gaskell,-P-C; Small,-G-W; Roses,-A-D; Haines,-J-L; Pericak-Vance,-M-A., Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families, (1993) Science. 261(5123): 921-3].
It is assumed that inhibition of the activity of β-secretase and γ-secretase would suppress the production of amyloid protein, leading to therapeutic drugs that could halt or slow the progress of Alzheimer's disease. While a reaction of a-secretase, in addition to β-secretase, is also involved in the first step of APP hydrolysis, inhibition of the reaction of γ-secretase in the second step is expected to have an effect with wider spectrum, because that reaction commonly follows either type of the first step reaction.
Though final identification of γ-secretase is yet to be reached, it is demonstrated that presenilin-1 is playing an important role. According to animal experiments or cell culture experiments, defunctionalization of presenilin-1 reportedly caused abnormalities in the development of cranial nerves or the formation of spinal column, or abnormalities in the development of lymphocytes. Thus, presenilin-1 is known to have a variety of functions in addition to the one relating the amyloid protein.
It must be taken account that nonspecific suppression of γ-secretase's activity could trigger some severe side effects such as induction of cancer [Hardy, J., Israel, A. Alzheimer's disease, In search of gamma-secretase, (1999) Nature. 398(6727) 466-7].
Presently known γ-secretase inhibitors are: inhibitors which are peptidomimetic compounds based on reports about the γ-site of the substrate APP, located at the carboxyl terminus of amyloid protein portion, on which the enzyme acts [Mori H., Takio K., Ogawara M. & Selkoe D. J. j, Mass spectrometry of purified amyloid b protein in Alzheimer's disease. (1992) J. Biol. Chem. 267, 17082-17086; Roher A. E. , Lowenson J. D., Clarke S., Wolkow C., Wang R., Cotter R. J., Reardon I. M., Zurcher-Neely H. A., Heinrikson R. L., Ball M. J., et al. Structural alterations in the peptide backbone of beta-amyloid core protein may account for its deposition and stability in Alzheimer's disease. (1993) J Biol Chem. 268(5), 3072-3083] and enzyme inhibitors based on reports pointing out the importance of aspartic acid active site [Wolfe M. S., Xia W., Ostaszewski B. L., Diehl T. S., Kimberly W. T., Selkoe D. J. Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity. (1999) Nature 398 (6727), 513-517].
As for a peptidomimetic compound, DFK-167 is known [Wolfe M. S., Citron M., Diehl T. S., Xia W., Donkor I. C. and Selkoe D. J.: A substrate-based difluoro ketone selectively inhibits Alzheimer's g-secretase activity. (1998) J. Med. Chem., 41(1), 6-9].
As for enzyme inhibitors, compounds screened from known inhibitors are reported. They are, L-685,458, which was made in the process of development of drugs for AIDS [Shearman, M. S., Beher, D., Clarke, E. E., Lewis, H. D., Harrison, T., Hunt, P., Nadin, A., Smith, A. L., Stevenson, G., Castro, J. L. L-685,458, an aspartyl protease transition state mimic, is a potent inhibitor of amyloid beta-protein precursor gamma-secretase activity. (2000) Biochemistry 39 (30), 8698-8704] and JLK-6, which is a α-chymotrypsin inhibitor [Nakajima, K., Powers, J. C., Ashe, B. M., Zimmerman, M. Mapping the extended substrate binding site of cathepsin G and human leukocyte elastase. Studies with peptide substrates related to the alpha 1-protease inhibitor reactive site. (1979) J. Biol. Chem. 254 (10), 4027-32].
Although the peptidomimetic compound-type inhibitors developed based on the γ-site of the substrate APP and the inhibitors developed based on the active site of the enzyme are all potent inhibitors of amyloid protein production, each has significant troubles. First, DFK-167 is an inhibitor that was designed for the γ-site and is a compound totally independent from the recent findings on the ε-site, thus it has a different target with regard to inhibitor designing. Inhibitors L-685,458 and JLK-6 are not compounds originally developed as specific inhibitors of γ-secretase involved in the production of the amyloid protein from the APP. Apart from the specificity problem as γ-secretase inhibitors, there are other problems concerning efficacy at the target tissue.